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eIF4A1 enhances LARP1-mediated translational repression during mTORC1 inhibition

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

Eukaryotic translation initiation factor (eIF)4A—a DEAD-box RNA-binding protein—plays an essential role in translation initiation. Recent reports have suggested helicase-dependent and helicase-independent functions for eIF4A, but the multifaceted roles of eIF4A have not been fully explored. Here we show that eIF4A1 enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA pulldown followed by sequencing revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTORC1 inhibition. This selective interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that deletion of EIF4A1 attenuated the translational repression of TOP mRNAs upon mTORC1 inactivation. Moreover, eIF4A1 increases the interaction between TOP mRNAs and LARP1 and, thus, ensures stronger translational repression upon mTORC1 inhibition. Our data show the multimodality of eIF4A1 in modulating protein synthesis through an inhibitory binding partner and provide a unique example of the repressive role of a universal translational activator.

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Fig. 1: eIF4A1 preferentially interacts with TOP mRNAs.
Fig. 2: LARP1 shows preferential binding to eIF4A1.
Fig. 3: Depletion of LARP1 eliminates the biased interaction between eIF4A1 and TOP mRNAs.
Fig. 4: EIF4A1-KO cells exhibit reduced translational repression of TOP mRNAs upon mTOR inhibition.
Fig. 5: eIF4A1 enhances the interaction between LARP1 and TOP RNAs.
Fig. 6: EIF4A1-KO cells are resistant to cytotoxicity mediated by mTOR inhibition.
Fig. 7: eIF4A2 is not required for the enhancement of LARP1-mediated translational repression.

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Data availability

The human genome hg38 and GENCODE release 32 annotations were obtained from UCSC Genome Browser (https://genome.ucsc.edu). The RNA pulldown-seq, ribosome profiling and RNA-seq (GSE184247) data used in this study were deposited in the National Center for Biotechnology Information. The MS (JPST002319) data used in this study were deposited in the Japan Proteome Standard Repository (jPOSTrepo). Source data are provided with this paper.

Code availability

Source codes of softwares for ribosome profiling data analysis were at GitHub (https://github.com/ingolia-lab/RiboSeq). The codes for data analysis of deep sequencing and MS have been deposited in Zenodo at https://doi.org/10.5281/zenodo.10644574 (ref. 69).

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Acknowledgements

We are grateful to all the members of the Iwasaki laboratory for constructive discussions, technical help and critical reading of the manuscript. We also thank K. Dodo and M. Sodeoka for the Pharos FX imaging scanner, the Support Unit for Bio-Material Analysis, the RIKEN CBS Research Resources Division for MS and Sanger sequencing and the HOKUSAI SailingShip supercomputer facility at RIKEN for computational support. Hippuristanol was a kind gift from J. Tanaka. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (JP20H05784 to S.I., JP21H05734 and JP23H04268 to Y.S. and JP21H05281 to T.I.); the Japan Society for the Promotion of Science (JP17H04998 to S.I., JP19J00920 and JP21K15023 to Y.S., JP19H03172 to T.I., JP18K14644 to K. Kashiwagi and JP20H03426 and JP20K21566 to K. Kuba); the Japan Agency for Medical Research and Development (JP20gm1410001 to S.I. and T.I. and JP23gm6910005 to Y.S.) and RIKEN (Pioneering Projects ‘Biology of Intracellular Environments’ to Y.S., S.I. and T.I.; the BDR Structural Cell Biology Project, the Pioneering Projects ‘Dynamic Structural Biology’ to T.I.; Special Postdoctoral Researchers to Y.S.; and Incentive Research Projects to Y.S.). The DNA libraries were sequenced by the Vincent J. Coates Genomics Sequencing Laboratory at the Universtiy of California Berkeley, which is supported by a National Institutes for Health Instrumentation Grant (S10 OD018174). Y.S. was a recipient of a Japan Society for the Promotion of Science Research Fellow (PD) and was supported by the RIKEN Special Postdoctoral Researchers Program.

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Authors and Affiliations

  1. RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan

    Yuichi Shichino, Mari Mito & Shintaro Iwasaki

  2. Department of Biochemistry and Metabolic Science, Akita University Graduate School of Medicine, Akita, Japan

    Tomokazu Yamaguchi & Keiji Kuba

  3. Department of Pharmacology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

    Tomokazu Yamaguchi & Keiji Kuba

  4. Laboratory for Translation Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan

    Kazuhiro Kashiwagi, Mari Takahashi & Takuhiro Ito

  5. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Nicholas T. Ingolia

  6. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan

    Shintaro Iwasaki

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  1. Yuichi Shichino
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Contributions

Conceptualization: Y.S. and S.I.; methodology: Y.S. and S.I.; formal analysis: Y.S. and T.Y.; investigation: Y.S., T.Y., M.M., K. Kashiwagi, M.T. and S.I.; resources: N.T.I.; writing—original draft: Y.S. and S.I.; writing—review and editing: Y.S., T.Y., M.M., K. Kashiwagi, M.T., T.I., N.T.I., K. Kuba and S.I.; visualization: Y.S.; supervision: T.I., N.T.I., K. Kuba and S.I.; funding acquisition: K. Kashiwagi, K. Kuba, T.I., Y.S. and S.I.

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Correspondence to Yuichi Shichino or Shintaro Iwasaki.

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Extended data

Extended Data Fig. 1 RNA pulldown-Seq with eIF4A1, Related to Fig. 1.

a, Western blotting of SBP-tagged eIF4A1 expressed in HEK293 cells. β-Actin was used as the loading control. b, CBB staining of purified SBP-eIF4A1 protein for RNA pulldown-Seq analysis.

Extended Data Fig. 2 Protein binding preference of eIF4A1, Related to Fig. 2.

a, Volcano plot of the heavy/light ratio. eIF4A1-bound proteins (defined as those with FDR < 0.05 and log2[heavy/light] > 1) are highlighted. b, Electropherogram of RNAs from the lysates used to obtain the data shown in Fig. 2c. c, Interactions between SBP-eIF4A1 and the series of N-terminal and C-terminal truncation mutants of LARP1. Naïve and SBP-EIF4A1 cells were transfected with 3 × FLAG-6 × His-tagged LARP1 and its variants. The domain structure of LARP1 is shown at the top.

Extended Data Fig. 3 Translational repression upon mTOR inhibition is attenuated in EIF4A1 KO cells, Related to Fig. 4.

a, Western blotting of eIF4A1 and eIF4A2 in the SBP-eIF4A1 (Phe163Leu-Ile199Met) eIF4A1SINI cell line. Cells were cultured in tetracycline-free medium. β-Actin was used as the loading control. b, Metagene plot of the 5′ ends of cytoplasmic footprints around start or stop codons in DMSO-treated naïve cells. RPM: reads per million mapped reads. c, Cumulative distribution of changes in the translation of all mRNAs and TOP mRNAs upon treatment with 10 µM PP242 in naïve cells. The p-value was calculated by the two-sided Mann-Whitney U test. d, Western blotting of LARP1, 4EBP1, and phosphorylated 4EBP1 (S65) upon 1 µM and 10 µM PP242 treatment. β-Actin was used as the loading control. e, Western blotting of RPTOR associated with 3 × FLAG-6 × His-tagged LARP1. Cells were subjected to amino acid starvation for 6 h. f, Relative luminescence produced by TOP reporters in naïve, EIF4A1 KO, and SBP-EIF4A2 cells treated with 150 nM Torin-1 for 2 h. The Renilla luciferase signal was normalized to that of firefly luciferase. Data from three replicates (points) and the mean values (bars) are shown. The p-values were calculated by the two-sided Tukey-Kramer multiple comparison test. Tet: tetracycline. g, Western blotting of LARP1, eIF4A1, and 4EBP1 upon the knockdown of LARP1. Cells were treated with 3 µM PP242 for 2 h. β-Actin was used as the loading control. h and i, Relative luminescence produced by the TOP (h) and TOPm (i) reporters in naïve and EIF4A1 KO cells upon the knockdown of LARP1. Cells were treated with 3 µM PP242 for 2 h. The Renilla luciferase signal was normalized to the firefly luciferase signal. Data from three replicates (points) and the mean values (bars) are shown. The p-values were calculated by the two-sided Tukey-Kramer multiple comparison test.

Extended Data Fig. 4 Translational repression upon mTOR inhibition is attenuated in EIF4A1 KO cells, Related to Fig. 4.

a, Global protein synthesis rate in EIF4A1 KO and SBP-EIF4A2 cells, as measured by OP-puro labeling. The quantification of nascent proteins (IRDye 800 signal) normalized to total protein (CBB signal) from three replicates (points) and the mean values (bars) are shown on the right. The p-values were calculated by two-sided Dunnett’s multiple comparison test. Tet: tetracycline. b, Relative viability of naïve, EIF4A1 KO, and SBP-EIF4A2 cells. Tetracycline (Tet) was added 48 h before measurement of luminescence. Data from three replicates (points) and the mean values (bars) are shown. The p-values were calculated by two-sided Tukey-Kramer multiple comparison test. c, Relative luminescence produced by TOP reporters in the indicated cells with the indicated treatment. For the drug treatment, cells were incubated with 0.1 µM hippuristanol for 2 h. The Renilla luciferase signal was normalized to that of firefly luciferase. Data (compared to those in naïve cells with DMSO treatment) from three replicates (points) and the mean values (bars) are shown. d, Relative luminescence produced by TOP reporters in the indicated cells with the indicated treatment. For the drug treatment, cells were incubated with 0.1 µM hippuristanol and/or 3 µM PP242 for 2 h. The Renilla luciferase signal was normalized to that of firefly luciferase. Data (compared to cells without PP242 treatment under each condition) from three replicates (points) and the mean values (bars) are shown. The p-values were calculated by the two-sided Tukey-Kramer multiple comparison test.

Extended Data Fig. 5 Translational repression upon amino acid starvation is attenuated in EIF4A1 KO cells, Related to Fig. 4.

a, Phosphorylation status of mTORC1 substrates (S6K1 and 4EBP1) and an mTORC2 substrate (AKT) in naïve cells upon amino acid starvation for 6 h. Cells were treated with PP242 or Torin-1 for 2 h. β-Actin was used as the loading control. b, Polysome analysis of naïve and EIF4A1 KO cells upon amino acid starvation for 6 h. c, Western blotting of LARP1, RPS6, and RPL11 proteins from subpolysome, light polysome, and heavy polysome fractions. d, Abundances of endogenous TOP mRNAs (RPL10 and RPL13) from subpolysome, light polysome, and heavy polysome fractions, as measured by RT-qPCR. The relative poly/subpoly ratio [(light+heavy)/sub] is shown on the right. The p-values were calculated by two-sided Welch’s t-test. e, Boxplot of changes in the levels of all mRNAs (n = 13,300 mRNAs) and TOP mRNAs (n = 93 mRNAs) upon EIF4A1 knockout. The medians (center line), upper/lower quartiles (box limits), and 1.5× interquartile ranges (whiskers) are shown. The p-values were calculated by the two-sided Mann-Whitney U test.

Extended Data Fig. 6 LARP1 protein purified from PP242-treated cells exhibits increased interaction for the TOP motif, Related to Fig. 5.

a, CBB staining of recombinant 3 × FLAG-6 × His-LARP1 purified from HEK293 cells treated with or without 10 µM PP242. b, (Top) An RNA crosslinking assay was performed to investigate the interactions between recombinant LARP1 and 0.4 µM cap-labeled TOP or TOPm RNAs. 3 × FLAG-6 × His-LARP1 purified from HEK293 cells treated with or without 10 µM PP242 was used. (Bottom) Input cap-labeled TOP or TOPm RNAs were spotted on the nylon membrane. c, SYPRO Ruby staining of recombinant 3 × FLAG-6 × His-LARP1 and LARP1(∆DM15) purified from HEK293 cells treated with 10 µM PP242. d, RNA crosslinking assay of the interaction between purified LARP1(∆DM15) protein and 0.4 µM cap-labeled TOP RNA. 3 × FLAG-6 × His-LARP1 and LARP1(∆DM15) purified from PP242-treated cells were used. e, CBB staining of recombinant 3 × FLAG-6 × His-LARP1 and LARP1(∆327-496) purified from HEK293 cells treated with 10 µM PP242. f, Western blotting of LARP1 associated with SBP-eIF4A1 proteins. Cells were subjected to amino acid starvation for 6 h. g, In vitro pulldown assay of the interactions between recombinant eIF4A1 and LARP1 purified from cells treated with or without 10 µM PP242. Unconjugated beads and beads conjugated to 3 × FLAG-6 × His-tagged LARP1 were incubated with 2 µM or 10 µM His-tagged eIF4A1. For the loading controls, 2 µM and 10 µM 6 × His-eIF4A1 protein were used. Proteins were stained using SYPRO Ruby.

Extended Data Fig. 7 The decrease in cell viability upon mTOR inhibition is attenuated in EIF4A1 KO cells, Related to Fig. 6.

a, Relative viability rate of naïve, EIF4A1 KO, and SBP-EIF4A2 cells treated with Torin-1 for 48 h. Tetracycline (Tet) was added simultaneously with Torin-1. Signals were normalized to those in DMSO-treated samples. Mean values and error bars indicating standard deviations (n = 3 independent replicates) are shown. The p-values for 0.04 µM Torin-1 were calculated by the two-sided Tukey-Kramer multiple comparison test. b, Relative viability rate of naïve, EIF4A1 KO, and SBP-EIF4A2 cells upon amino acid starvation for 30 h. Signals were normalized to those in nonstarved samples. Data from three replicates (points) and the mean values (bars) are shown. The p-values were calculated by the Tukey-Kramer multiple comparison test. Tet: tetracycline. c, Change in the cell numbers of Eif4a1 KO MEFs. A total of 1 × 104 cells were incubated with 1 µM tamoxifen for 24 h and were then cultured in medium without tamoxifen for the indicated time. Mean values and error bars indicating standard deviations (n = 3 independent replicates) are shown. The p-values at 4 days were calculated by two-sided Welch’s t-test.

Extended Data Fig. 8 Characterization of EIF4A2 KO cells, Related to Fig. 7.

a, CBB staining of purified SBP-eIF4A2 protein for RNA pulldown-Seq analysis. b, Electropherogram of RNAs from the lysates used to obtain the data shown in Fig. 7b. c, Schematic of gRNAs designed for EIF4A2 gene knockout and mutation mediated by CRISPR/Cas9 gene editing. d, Western blotting of eIF4A1, eIF4A2, eIF4G1, and eIF4E1 in naïve, EIF4A1 KO, and EIF4A2 KO cells. β-Actin was used as the loading control. e, Global protein synthesis rate in EIF4A1 KO and EIF4A2 KO cells, as measured by OP-puro labeling. The quantification of nascent proteins (IRDye 800 signal) normalized to total protein (CBB signal) in three replicates (points) and the mean values (bars) are shown on the right. The p-values were calculated by two-sided Dunnett’s multiple comparison test. f, Viability of naïve, EIF4A1 KO, and EIF4A2 KO cells. Mean values and error bars indicating standard deviations (n = 3 independent replicates) are shown.

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Supplementary Tables 1–7 with results of RNA pulldown-seq, proteomics, ribosome profiling, RNA-seq and reporter assays.

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Shichino, Y., Yamaguchi, T., Kashiwagi, K. et al. eIF4A1 enhances LARP1-mediated translational repression during mTORC1 inhibition. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01321-7

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